The barcoding of cellular mRNAs with unique identifiers, such as incorporation of unique nucleotide sequences during cDNA synthesis, is widely used in genomic applications (A. E. Saliba, A. J. Westermann, S. A. Gorski, J. Vogel, Single-cell RNA-seq: advances and future challenges. Nucleic Acids Research 42, 8845 (2014)). For example, it can be exploited in single-cell analysis to clearly assign specific mRNA sequences to individual cells, even when sequencing pooled samples. Furthermore, genetic barcoding is of major use for pharmacogenomics studies, e.g. for analysing the effect of a particular drug on the transcriptome. This approach holds enormous potential in drug screening, since it enables screening drug candidates not only for the effect on one particular drug target, but rather for their effect on global gene expression. In consequence, this helps revealing potential side effects, but as well allows to predict the effect of drug combinations and to establish “precision therapies”.
Current high throughout cell barcoding approaches make use of beads or gel particles displaying thousands of copies of the same barcoded polyT primer (H. C. Fan, G. K. Fu, S. P. A. Fodor, Combinatorial labeling of single cells for gene expression cytometry. Science 347, 628, Feb. 6, 2015; E. Z. Macosko, A. Basu, R. Satija, J. Nemesh, K. Shekhar, M. Goldman, I. Tirosh, A. R. Bialas, N. Kamitaki, E. M. Martersteck, J. J. Trombetta, D. A. Weitz, J. R. Sanes, A. K. Shalek, A. Regev and S. A. McCarroll, Cell, 2015, 161, 1202-1214). Each bead displays a different primer, however their exact barcode sequence is not known since they are synthesized in a combinatorial split-and-pool (solid phase) synthesis. The beads are then incubated with the cell samples (optionally on the single-cell level), and the cellular mRNAs are reverse transcribed into cDNA harbouring the respective barcode sequences. This allows pooling the cells for sequencing their transcriptome, while still being able to distinguish transcriptome patterns from different cells. Nonetheless, a direct coupling of a particular transcriptome pattern with a particular individual cell is impossible.
Overcoming this limitation, the inventors have developed a microfluidic approach enabling to barcode single cells, as well as cell populations. The method is based on the combinatorial assembly of barcoding primers, using valve-based microfluidic technology. In contrast to existing systems (e.g. as sold by Fluidigm), as well as recent publications, for example in Science (H. C. Fan, G. K. Fu, S. P. A. Fodor, Combinatorial labeling of single cells for gene expression cytometry. Science 347, 628, Feb. 6, 2015; E. Z. Macosko, A. Basu, R. Satija, J. Nemesh, K. Shekhar, M. Goldman, I. Tirosh, A. R. Bialas, N. Kamitaki, E. M. Martersteck, J. J. Trombetta, D. A. Weitz, J. R. Sanes, A. K. Shalek, A. Regev and S. A. McCarroll, Cell, 2015, 161, 1202-1214; A. M. Klein, L. Mazutis, I. Akartuna, N. Tallapragada, A. Veres, V. Li, L. Peshkin, D. A. Weitz and M. W. Kirschner, Cell, 2015, 161, 1187-1201), this technology is not making use of degenerated, random barcodes, but rather allows labelling cells with barcodes of known identity. Hence one cannot only distinguish between the transcriptome of different cells, but also correlate a particular transcriptome with a particular phenotype or a sample exposed to a particular drug. This opens the way for manifold new applications.